Progress in FY2019 was in the following areas: EFFECT OF ASP-TO-VAL MUTATION ON HNRNPA2 FIBRIL FORMATION: Previous work by other labs has shown that an Asp-to-Val mutation at residue 290 of the low-complexity domain of hnRNPA2 leads to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) by facilitating intracellular hnRNPA2 aggregation. To understand the structural basis for this phenomenon, we performed solid state NMR and electron microscopy measurements on fibrils formed by wild-type hnRNPA2-LC and by the D290V mutant. The data indicate that residue 290 is near the end of a beta-strand that forms parallel beta-sheets in the wild-type fibrils, and that Asp290 is negatively charged in the fibrils near neutral pH. This suggests that replacement of Asp290 by Val eliminates intermolecular electrostatic repulsions that make wild-type hnRNPA2-LC fibrils less stable, and creates new hydrophobic interactions that may stabilize the D290V mutant fibrils. In fact, from guanidine denaturation experiments, we find that wild-type fibrils are indeed less stable than mutant fibrils by approximately 1.2 kJ/mol. A paper describing these results was published in early FY2019 (Murray et al., PNAS 2018) MOLECULAR STRUCTURE OF C-TERMINAL FUS-LC FIBRIL CORE: In earlier work (Murray et al., Cell 2017), we showed that fibrils formed by the 214-residue low-complexity domain of the FUS protein have a cross-beta, rigidified core that consists of residues 39-95. The C-terminal half of the FUS-LC sequence was found to be dynamically disordered, such that NMR signals from the C-terminal half could be observed with solution NMR techniques and exhibited NMR chemical shifts close to random-coil values. Surprisingly, however, the amino compositions of the C-terminal and N-terminal halves of the FUS-LC sequence are nearly the same, consisting primarily of Gly, Ser, Gln, and Tyr residues. It is therefore unclear why the N-terminal half becomes rigid and structurally ordered, while the C-terminal half remains flexible and disordered in the context of the full-length sequence. Subsequently, we have found that the C-terminal half alone (residues 111-214) forms highly ordered fibrils, which are apparently monomorphic based on electron microscopy. We are now investigating the molecular structure of these C-terminal FUS-LC fibrils, using both solid state NMR and cryo-EM techniques. A large set of 2D and 3D solid state NMR spectra have been obtained. Tentative site-specific chemical shift assignments have been obtained. Segmentally isotopically labeled samples are being prepared to confirm these assignments. High-quality cryo-EM images have also been obtained, and are currently being analyzed. We expect to complete this structure determination in FY2020.